Induction of hematopoietic cellular chimerism through bone marrow transplantation can lead to life-long transplantation tolerance without the need for chronic immunosuppression. However, bone marrow transplantation across major histocompatibility (MHC) barriers is severely limited by problems associated with the severity of the host preparative regimen required to achieve engraftment, the potential for lethal graft-versus-host disease (GVHD), and a high rate of engraftment failure. These problems could potentially be avoided by using a gene therapy approach to introduce allogeneic MHC genes into autologous bone marrow resulting in a state of molecular rather than cellular chimerism. In the last funding period, we examined whether such a gene therapy based approach could be used to induce tolerance to an allogeneic major histocompatibility (MHC) class I antigen. Reconstitution of lethally irradiated B10.AKM mice (H-2Kk, Ik, Dq) with syngeneic bone marrow infected with retroviruses carrying the allogeneic MHC class I gene H-2Kb, resulted in life-long expression of Kb on the surface of bone marrow cells, and specific tolerance to Kb. Mice reconstituted with H-2Kb transduced bone marrow accepted K b disparate skin allografts long-term without the need for any additional immunosuppression. Thus, genetic engineering of bone marrow using retroviral transduction to establish molecular chimerism can be used to induce long-term stable tolerance to transplantation antigens, effectively re-shaping the immunological repertoire. The specific aims of this proposal are: 1) To determine the mechanism by which CD4 T cell tolerance induced by gene therapy is maintained in molecular chimeras; 2) Develop clinically relevant non-myeloablative protocols to induce tolerance by genetic engineering of bone marrow; and 3) Test the hypothesis that induction of molecular chimerism can be used to induce tolerance to MHC class I and class II disparate grafts and prevent acute as well as chronic allograft rejection. These studies should expand the use of gene therapy to induce immunological tolerance, provide practical information important for the field of transplantation on mechanisms of tolerance, and may also advance our understanding of self-nonself discrimination with respect to MHC antigens. Ultimately, these studied may provide a novel means of inducing acceptance of allogeneic organ transplants without the need for life-long immunosuppression.